We propose to continue our investigations into the quantitative relationships between the number of H ion translocated out of respiring mitochondrial and the number of electrons flowing down the respiratory chain to an atom of oxygen i.e., the H ion/O ratio. We propose to carry out further measurements with new methods we have developed, in order to confirm our finding that as many as 12 H ion are ejected per pair of electrons reducing an atom of oxygen. We also propose to determine the specific electron carriers in the mitochondrial respiratory chain which are responsible for translocating H ion and which thus enable the transduction of oxidation-reduction energy into an electrochemical gradient across the mitochondrial membrane. In particular, we propose to examine closely energy conserving site 3, promoted by cytochrome oxidase, since our investigations indicate that this is the key enzyme in the respiratory chain which is responsible for regulating the rate of H ion extrusion. The kinetics, H ion stoichiometry, and dependence of activity on oxygen tension in the cytochrome oxidase reaction will be examined in the light of the physiological aspects of tissue function. We also propose to examine the membrane transport mechanisms for ATP, ADP, and phosphate during ATP-dependent reverse electron flow, an important regulatory process in the correlation of respiration and glycolysis. Finally, we also propose to investigate the mechanism of H ion "pumping" coupled to electron transport and to ATP synthesis in mitochondria.